1. Field of the Invention
The present invention relates to a bio-assay method, and particularly to a method for direct detection of lipid binding agents in membrane to quantitatively detect cell membrane active agents (MAAs) directly in lipid membranes.
2. Description of the Related Art
Membrane proteins (MPs) in a cell represent about 1% of the cellular proteins, but 50% of the cellular constituent targeted drugs end up acting on MPs. Whenever membrane is approached by agents, some of which physically reside in the vicinity (e.g., MPs), while others get dragged from external sources (e.g., drugs), the membrane's main constituent lipids experience a state of physical coexistence with the agents. The agents are found to perform various physiological activities due to their membrane-bound coexistence. Based on their specific or nonspecific actions upon membrane, we define them as a class or various classes of membrane active agents (MAAs). Theoretical, computational, and experimental techniques may be used to understand the membrane-targeted activities through investigating complex structures resulting from MAA-lipid coexistences. The lipid bilayer hosted ion channels due to MAAs, e.g., MPs or antimicrobial peptides (AMPs), are examples of theoretically understandable and experimentally detectable events. The MAA-induced events, and occasionally lipid reorganization, also creates specific stable channel structures in the membranes. For example, ceramide is found to induce lipid channels.
The geometry of cluster structure also is found to be MAA specific. In recent investigations, small DNA oligos that are called ‘aptamers’ are found to show physical lipid-specific liposome binding properties, and thus perhaps appear with properties that make them includable in the class of MAAs.
Use of membrane active agents (MAAs), such as peptides, aptamers, general biomolecules, small proteins, detergents, etc., is a common practice among scientists for various purposes, such as structure analysis, disease research, drug discovery research, etc. Also, the structural aspects of MAA-lipid complexes, along with their phenomenological statistics and dynamics, are quite known. However, quantitative analysis to pinpoint mole fraction binding of MAAs with membrane, the energetics behind binding, and molecular mechanisms to determine the quantitative binding probability are either not available or limited, to date. Besides, the underlying molecular mechanisms are left either unexplored or addressed poorly using apparently a few incorrect hypotheses, approaches, and formalisms. Therefore, it would be desirable to develop a direct detection method (DDM) to detect cell membrane active agents (MAAs) quantitatively directly in lipid membrane and thereby develop a method that could act as a ‘universal platform’ to test and certify an agent as a candidate with measurable probability of interaction or binding with a target structure in a biological system.
Thus, a method for direct detection of lipid binding agents in membrane solving the aforementioned problems is desired.